The importance of glycosylation in many biological processes is commonly accepted and has been discussed in detail throughout the literature over the last 30 years. Glycosylation is a common and highly diverse post-translational modification of proteins in eukaryotic cells. Various cellular processes have been described, involving carbohydrates on the protein surface. The importance of glycans in protein stability, protein folding and protease resistance have been demonstrated in the literature. In addition, the role of glycans in cellular signalling, regulation and developmental processes has been demonstrated in the art.
The oligosaccharides are mainly attached to the protein backbone, either by N-(via Asn) or O-(via Ser-Thr) glycosidic bonds, whereas N-glycosylation represents the more common type found in glycoproteins. Variations in glycosylation site occupancy (macroheterogeneity), as well as variations in these complex sugar residues attached to one glycosylation site (microheterogeneity) result in a set of different protein glycoforms. These have different physical and biochemical properties, which results in additional functional diversity. In manufacturing of therapeutic proteins in mammalian cell cultures, macro- and microheterogeneity were shown to affect properties like protein solubility, structural stability, protease resistance, or biological and clinical activity, see for example Butler, M., Cytotechnology, 2006, 50, 57-76. For instance, the relevance of the glycosylation profile for the therapeutic profile of monoclonal antibodies is well documented; see e.g. Parekh, et al., Nature, 1985, 316, 452-457.
Glycan biosynthesis is a non-template-driven process, involving the cell glycosylation machinery. N-glycan structures are also depending on various factors during the production process, like substrates levels and other culture conditions. Thus, the glycoprotein manufacturing does not only depend on the glycosylation machinery of the host cell but also on external parameters, like cultivation conditions and the extracellular environment. Culture parameters affecting glycosylation include temperature, pH, aeration, supply of substrates or accumulation of byproducts such as ammonia and lactate. In case of recombinant glycoprotein or antibody manufacturing, characterization of glycosylation profiles attracts increasing interest. In particular, because of regulatory reasons, the glycosylation profile of drugs has to be determined.
Today, complex soluble but also oligomeric and/or polymeric carbohydrate mixtures, obtained synthetically or from natural sources, like plants or human or animal milk are used as nutrition additives or in pharmaceuticals. The occurrence of sialic acids or sialic acid derivatives and the occurrence of monosaccharides having a phosphate, sulphate or carboxyl group within those complex natural carbohydrates is even increasing their complexity. Because of this complexity, those prebiotic oligo- or polysaccharides, like neutral or acidic galacto-oligosaccharides, long chain fructo-oligosaccharides, which can have nutritional and/or biological effects, are gaining increasing interest for food and pharmaceutic industry.
A wide range of strategies and analytical techniques for analysing glycoproteins, glycopeptides and released N-glycans or O-glycans have been established including e. g 2D-HPLC profiling, mass spectrometry and lectin affinity chromatography, as reviewed by Geyer and Geyer, Biochimica Et Biophysica Acta-Proteins and Proteomics 2006, 1764, 1853-1869 and Domann et al., Practical Proteomics, 2007, 2, 70-76.
To obtain structural data of complex molecules, today carbohydrates are either analysed by mass spectrometry (MS) or nuclear magnetic resonance spectroscopy (NMR) which are generally laborious and time consuming techniques regarding sample preparation and data interpretation.
Each of these techniques has advantages as well as drawbacks. Choosing one, respectively a set of these methods for a given problem can become a time- and labor-intensive task. For example, NMR provides detailed structural information, but is a relatively insensitive method (nmol), which can not be used as a high-throughput method. Using MS is more sensitive (fmol) than NMR. However, quantification can be difficult and only unspecific structural information can be obtained without addressing linkages of monomeric sugar compounds. Both techniques require extensive sample preparation and also fractionation of complex glycan mixtures before analysis to allow evaluation of the corresponding spectra. Furthermore, a staff of highly skilled scientists is required to ensure that these two techniques can be performed properly.
Although separation techniques based on the capillary electrophoresis principle, like capillary gel electrophoresis where considered for complex carbohydrate separation in the art before, e.g Callewaert, N. et al, Glycobiology 2001, 11, 275-281, WO 01/92890, Callewaert, N. et al, Nat Med. 2004, 10 429-434, there is still an ongoing need for a reliable and fast system allowing automated high throughput carbohydrate analysis.
As identified by Domann et al, normal phase chromatography and capillary gel electrophoresis have an excellent selectivity for the analysis of fluorescently labelled glycans. Serious drawbacks regarding the limit of detection and the linear dynamic range of CGE-LIF compared to normal phase liquid chromatography with fluorescence detection have been reported. Furthermore with respect to CGE-LIF no methods are described in the art allowing to rapidly monitor alterations in carbohydrate mixture composition patterns (e.g. glycosylation patterns) including fast and straightforward structure elucidation, without the need for complex data evaluation. Further, there is a need in the art to provide means and methods allowing for determination and identification of carbohydrate mixtures of unknown composition enabling identification of the carbohydrate structures. In particular, there is a need for a sensitive but reproducible and robust system and method allowing the identification or determination of carbohydrate mixture composition patterns (e.g.: glycosylation patterns) as well as of carbohydrate compositions of unknown constitution in automated high throughput mode. In particular, for the latter, the method and system must ensure very accurate and reproducible analysis of carbohydrates whereby said analysis is essentially independent from sample type and origin, timepoint of analysis, laboratory, instrument and operator.